Acidic Insulin Preparations Having Improved Stability

ABSTRACT

The invention relates to a pharmaceutical formulation comprising a polypeptide selected from the group consisting of insulin, an insulin metabolite, an insulin analog, an insulin derivative and combinations thereof; a surfactant or combinations of two or more surfactants; optionally a preservative or combinations of two or more preservatives; and optionally an isotonicizing agent, buffers or further excipients or combinations thereof, the pharmaceutical formulation having a pH in the acidic range.

This application is a continuation of U.S. application Ser. No.12/328,208, filed Dec. 4, 2008, now U.S. Pat. No. 7,713,930, issued, May11, 2010, which is a continuation of U.S. application Ser. No.11/089,777, filed Mar. 25, 2005, now U.S. Pat. No. 7,476,652, issued,Jan. 13, 2009, which is a continuation of Ser. No. 10/461,740, filedJun. 13, 2003, now abandoned; all of which are incorporated herein byreference in their entirety which claims the benefit of U.S. ProvisionalApplication No. 60/409,338, filed Sep. 9, 2002, and Federal Republic ofGermany Application 10227232.8, filed Jun. 18, 2002.

SUMMARY OF THE INVENTION

The invention relates to a pharmaceutical formulation comprising apolypeptide selected from the group consisting of insulin, an insulinmetabolite, an insulin analog, an insulin derivative or combinationsthereof; a surfactant or combinations of two or more surfactants;optionally a preservative or combinations of two or more preservatives;and optionally an isotonicizing agent, buffers or further excipients orcombinations thereof, the pharmaceutical formulation having a pH in theacidic range. These formulations can be employed for the treatment ofdiabetes, and are particularly suitable for preparations in which a highstability to thermal and/or physicomechanical stress is necessary. Theinvention likewise relates to parenteral preparations which contain suchformulations and can be used in diabetes and to methods for producingthe preparations and for improving the stability of insulinpreparations.

BACKGROUND OF THE INVENTION

Worldwide, approximately 120 million people suffer from diabetesmellitus. Among these, approximately 12 million are type I diabetics,for whom the substitution of the lacking endocrine insulin secretion isthe only currently possible therapy. The affected persons are dependentlifelong on insulin injections, as a rule a number of times daily. Incontrast to type I diabetes, there is not basically a deficiency ofinsulin in type II diabetes, but in a large number of cases, especiallyin the advanced stage, treatment with insulin, optionally in combinationwith an oral antidiabetic, is regarded as the most favorable form oftherapy.

In the healthy person, the release of insulin by the pancreas isstrictly coupled to the concentration of blood glucose. Elevated bloodglucose levels, such as occur after meals, are rapidly compensated by acorresponding increase in insulin secretion. In the fasting state, theplasma insulin level falls to a basal value which is adequate toguarantee a continuous supply of insulin-sensitive organs and tissuewith glucose and to keep hepatic glucose production low at night. Thereplacement of endogenous insulin secretion by exogenous, mostlysubcutaneous administration of insulin, as a rule does not approximatethe quality of the physiological regulation of the blood glucosedescribed above. Often, deviations of blood glucose upward or downwardoccur, which in their severest forms can be life-threatening. Inaddition, however, blood glucose levels which are increased for yearswithout initial symptoms are a considerable health risk. The large-scaleDCCT study in the USA (The Diabetes Control and Complications TrialResearch Group (1993) N. Engl. J. Med. 329, 977-986) demonstratedclearly that chronically elevated blood glucose levels are essentiallyresponsible for the development of diabetic late damage. Diabetic latedamage is microvascular and macrovascular damage which is manifested,under certain circumstances, as retinopathy, nephropathy or neuropathyand leads to loss of sight, kidney failure and the loss of extremitiesand is moreover accompanied by an increased risk of cardiovasculardiseases. In view of this, an improved therapy of diabetes should beaimed at keeping the blood glucose as closely as possible in thephysiological range. According to the concept of intensified insulintherapy, this should be achieved by repeated daily injections of rapid-and slow-acting insulin preparations. Rapid-acting formulations aregiven at meals in order to level out the postprandial increase in theblood glucose. Slow-acting basal insulins should ensure the basic supplywith insulin, in particular during the night, without leading tohypoglycemia.

Insulin is a polypeptide of 51 amino acids, which are divided into 2amino acid chains: the A chain having 21 amino acids and the B chainhaving 30 amino acids. The chains are connected to one another by meansof 2 disulfide bridges. Insulin preparations have been employed fordiabetes therapy for many years. Not only are naturally occurringinsulins used, but recently also insulin derivatives and analogs.

Insulin analogs are analogs of naturally occurring insulins, namelyhuman insulin or animal insulins, which differ by substitution of atleast one naturally occurring amino acid residue with other amino acidsand/or addition/removal of at least one amino acid residue from thecorresponding, otherwise identical, naturally occurring insulin. Theamino acids can in this case also be those which do not occur naturally.

Insulin derivatives are derivatives of naturally occurring insulin or aninsulin analog which are obtained by chemical modification. Thischemical modification can consist, for example, of the addition of oneor more specific chemical groups to one or more amino acids. As a rule,insulin derivatives and insulin analogs have a somewhat modified actioncompared with human insulin.

Insulin analogs having an accelerated onset of action are described inEP 0 214 826, EP 0 375 437 and EP 0 678 522. EP 0 124 826 relates, interalia, to substitutions of B27 and B28. EP 0 678 522 describes insulinanalogs which in position B29 have various amino acids, preferablyproline, but not glutamic acid. EP 0 375 437 includes insulin analogswith lysine or arginine in B28, which can optionally be additionallymodified in B3 and/or A21.

In EP 0 419 504, insulin analogs are disclosed which are protectedagainst chemical modifications, in which asparagine in B3 and at leastone further amino acid in the positions A5, A15, A18 or A21 aremodified.

In WO 92/00321, insulin analogs are described in which at least oneamino acid of the positions B1-B6 is replaced by lysine or arginine.According to WO 92/00321, insulins of this type have a prolonged action.The insulin analogs described in EP-A 0 368 187 also have a delayedaction.

The insulin preparations of naturally occurring insulins on the marketfor insulin substitution differ in the origin of the insulin (e.g.bovine, porcine, human insulin), and also the composition, whereby theprofile of action (onset of action and duration of action) can beinfluenced. By combination of various insulin preparations, verydifferent profiles of action can be obtained and blood sugar valueswhich are as physiological as possible can be established. RecombinantDNA technology today makes possible the preparation of such modifiedinsulins. These include insulin glargine(Gly(A21)-Arg(B31)-Arg(B32)-human insulin) with a prolonged duration ofaction. Insulin glargine is injected as an acidic, clear solution andprecipitates on account of its solution properties in the physiologicalpH range of the subcutaneous tissue as a stable hexamer associate.Insulin glargine is injected once daily and is distinguished comparedwith other long-acting insulins by its flat serum profile and thereduction of the danger of nightly hypoglycemia associated therewith(Schubert-Zsilavecz et al., 2:125-130 (2001)).

The specific preparation of insulin glargine, which leads to theprolonged duration of action, is characterized, in contrast topreviously described preparations, by a clear solution having an acidicpH. Especially at acidic pH, insulins, however, show a decreasedstability and an increased proneness to aggregation on thermal andphysicomechanical stress, which can make itself felt in the form ofturbidity and precipitation (particle formation) (Brange et al., J. Ph.Sci 86:517-525 (1997)).

The proneness to aggregation can additionally be promoted by hydrophobicsurfaces which are in contact with the solution (Sluzky et al., Proc.Natl. Acad. Sci. 88:9377-9381 (1991). Surfaces which can be consideredas hydrophobic are the glass vessels of the preparations, the stoppermaterial of the sealing caps or the boundary surface of the solutionwith the air supernatant. In addition, very fine silicone oil dropletscan function as additional hydrophobic aggregation nuclei in the takingof the daily insulin dose by means of customary, siliconized insulinsyringes and accelerate the process.

WO 01/43762 describes aqueous, parenteral pharmaceutical preparationscomprising a polypeptide and glycerol, in which the stabilization of thepreparation is to be achieved by purifying off destabilizingconstituents of the glycerol.

WO 00/23098 describes insulin preparations stabilized using polysorbate20 or poloxamer 188 for pulmonary administration, but does not describethe stabilization in an acidic solution against aggregation nuclei.

WO 02/076495 describes zinc-free and low-zinc insulin preparationshaving improved stability at room and body temperature and to mechanicalstress by the addition of surfactants, but does not describe thestabilization of acidic insulin preparations against hydrophobicaggregation nuclei.

The present invention was thus based on the object of findingpreparations for acid-soluble insulins containing surfactants, which aredistinguished by a high long-term stability to stress due to temperatureor physicomechanical stressing and tolerate a high stress withhydrophobic aggregation nuclei.

DETAILED DESCRIPTION OF THE INVENTION

It has now surprisingly been found that the addition of surfactants cangreatly increase the stability of acidic insulin preparations and thuspreparations can be produced which guarantee superior stability tohydrophobic aggregation nuclei for several months under temperaturestress.

The pharmaceutical preparations of the present invention contain 60-6000nmol/ml, preferably 240-3000 nmol/ml, of an insulin, an insulinmetabolite, an insulin analog or an insulin derivative.

The surfactants which can be used are, inter alia, nonionic surfactants.In particular, pharmaceutically customary surfactants are preferred,such as, for example: partial and fatty acid esters and ethers ofpolyhydric alcohols such as of glycerol, sorbitol and the like (SPAN®,TWEEN®, in particular TWEEN® 20 and TWEEN® 80, MYRJ®, BRIJ®), CREMOPHOR®or poloxamers. The surfactants are present in the pharmaceuticalcomposition in a concentration of 5-200 μg/ml, preferably of 5-120 μg/mland particularly preferably of 20-75 μg/ml.

The preparation can additionally optionally contain preservatives (e.g.phenol, cresol, parabens), isotonicizing agents (e.g. mannitol,sorbitol, lactose, dextrose, trehalose, sodium chloride, glycerol),buffer substances, salts, acids and alkalis and also further excipients.These substances can in each case be present individually oralternatively as mixtures.

Glycerol, dextrose, lactose, sorbitol and mannitol are customarilypresent in the pharmaceutical preparation in a concentration of 100-250mM, NaCl in a concentration of up to 150 mM. Buffer substances, such as,for example, phosphate, acetate, citrate, arginine, glycylglycine orTRIS (i.e. 2-amino-2-hydroxymethyl-1,3-propanediol) buffer andcorresponding salts, are present in a concentration of 5-250 mM,preferably 10-100 mM. Further excipients can be, inter alia, salts orarginine.

The invention therefore relates to a pharmaceutical formulationcomprising a polypeptide selected from the group consisting of insulin,an insulin analog, an insulin derivative, an active insulin metaboliteand combinations thereof; a surfactant or combinations of two or moresurfactants; optionally a preservative or combinations of two or morepreservatives; and optionally an isotonicizing agent, buffer substancesand/or further excipients or combinations thereof, the pharmaceuticalformulation being a clear solution which has a pH in the acidic range(pH 1-6.8), preferably pH 3.5-6.8, very particularly preferably 3.5-4.5.

Preferred pharmaceutical formulations of the present invention are thosewherein the surfactant is selected from the group consisting of partialand fatty acid esters and ethers of polyhydric alcohols such as ofglycerol and sorbitol, and polyols; the partial and fatty acid estersand ethers of glycerol and sorbitol being selected from the groupconsisting of SPAN®, TWEEN®, MYRJ®, BRIJ®, CREMOPHOR®; the polyols beingselected from the group consisting of polypropylene glycols,polyethylene glycols, poloxamers, PLURONICS®, and TETRONICS®; thepreservative being selected from the group consisting of phenol, cresol,and parabens; the isotonicizing agent being selected from the groupconsisting of mannitol, sorbitol, sodium chloride, and glycerol; theexcipients being selected from the group consisting of buffersubstances, acids, and alkalis; the insulin analog being selected fromthe group consisting of Gly(A21)-Arg(B31)-Arg(B32)-human insulin;Lys(B3)-Glu(B29)-human insulin; Lys^(B28)Pro^(B29) human insulin, B28Asp-human insulin, human insulin in which proline in position B28 hasbeen substituted by Asp, Lys, Leu, Val or Ala and where in position B29Lys can be substituted by Pro; AlaB26-human insulin; des(B28-B30)-humaninsulin; des(B27)-human insulin and des(B30)-human insulin; the insulinderivative being selected from the group consisting ofB29-N-myristoyl-des(B30) human insulin, B29-N-palmitoyl-des(B30) humaninsulin, B29-N-myristoyl human insulin, B29-N-palmitoyl human insulin,B28-N-myristoyl Lys^(B28)Pro^(B29) human insulin,B28-N-palmitoyl-Lys^(B28)Pro^(B29) human insulin,B30-N-myristoyl-Thr^(B29)Lys^(B30) human insulin,B30-N-palmitoyl-Thr^(B29)Lys^(B30) human insulin,B29-N-(N-palmitoyl-γ-glutamyl)-des(B30) human insulin,B29-N-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin,B29-N-(ω-carboxyheptadecanoyl)-des(B30) human insulin andB29-N-(ω-carboxyheptadecanoyl) human insulin.

A further subject of the invention is a pharmaceutical formulation suchas described above, in which the insulin, the insulin analog, the activeinsulin metabolite and/or the insulin derivative is present in aconcentration of 60-6000 nmol/ml, preferably in a concentration of240-3000 nmol/ml (this corresponds approximately to a concentration of1.4-35 mg/ml or 40-500 units/ml);

in which the surfactant is present in a concentration of 5-200 μg/ml,preferably of 5-120 μg/ml and particularly preferably of 20-75 μg/ml.

A further subject of the invention is a pharmaceutical formulation suchas mentioned above, in which glycerol and/or mannitol is present in aconcentration of 100-250 mM, and/or NaCl is preferably present in aconcentration of up to 150 mM.

A further subject of the invention is a pharmaceutical formulation suchas mentioned above, in which a buffer substance is present in aconcentration of 5-250 mM.

A further subject of the invention is a pharmaceutical insulinformulation which contains further additives such as, for example, saltswhich delay the release of insulin. Mixtures of such delayed-releaseinsulins with formulations described above are included therein.

A further subject of the invention is a method for the production ofsuch pharmaceutical formulations. Likewise, a further subject of theinvention is the use of such formulations for the treatment of diabetesmellitus.

A further subject of the invention is the use or the addition ofsurfactants as stabilizer during the process for the production ofinsulin, insulin analogs or insulin derivatives or their preparations.

EXAMPLES

The following examples illustrate, but by no means limit, the presentinvention.

Comparison investigations: Different preparations containing the insulinanalog insulin glargine (Gly(A21), Arg(B31), Arg(B32)-human insulin) areprepared. To this end, insulin glargine is suspended in one part ofwater for injection, dissolved at pH 3-4, the other constituents areadded, the pH is adjusted to 4.0+/−0.2 using hydrochloric acid/NaOH andthe mixture is made up to the final volume. The concentration of insulinglargine in each of the experiments described below is 3.6378 mg/ml(corresponds to 100 units/ml). A second preparation is producedidentically, but a specific amount of a surfactant is additionallyadded. The solutions are filled into 10 ml glass vessels (vials) andfitted with crimp caps. These vessels are now exposed to simulated inuse or physicomechanical stress conditions:

-   1. In use test: The vessels are sorted into boxes with turned-up    lids and stored during the investigation period of 28 days at    +25° C. and controlled room humidity with exclusion of light. To    simulate taking by the patient, once daily about 5 IU of the    solutions are withdrawn using a customary insulin syringe and    discarded. At the beginning and end of the working week this    procedure is carried out twice in order to simulate taking at the    weekend. Before each withdrawal, visual assessment of the solution    in the vessels for turbidity and/or particle formation is carried    out.-   2. Shaking test: The vessels are placed in a box with a turned-up    lid lying on a laboratory shaker having an incubator and thermostat    and shaken at 25° C. with 90 movements/min parallel to the    horizontal movement for a period of time of 10 days. After defined    times, the turbidity value of the samples is determined by means of    a laboratory turbidity photometer (nephelometer) in formaldazine    nephelometric units (formaldazine nephelometric unit=FNU). The    turbidity value corresponds to the intensity of the scattered    radiation of the light incident on suspended particles in the    sample.

Example 1 Stabilization of the in Use Period of Insulin Glargine UsingPolysorbate 20 (Tween® 20)

a) The solution is sterile-filtered through a combination of 0.2 μm and0.1 μm filters. It is then poured into 10 ml injection vials and sealedusing crimp caps having an inserted sealing disk.b) A comparison solution is prepared identically, but first a suitableamount of surfactant (10-30 ppm of polysorbate 20) is suspended in waterfor injection. The samples are stored at +5° C., 25° C. and 37° C. for afixed period of time.

10 samples in each case are then subjected to an in use test. Theresults are shown in the table below.

Storage for 3 Months at 5° C.

Number of vials with particle formation after Test sample 7 days 14 days21 days 28 days Insulin glargine 7 10 10 10 Insulin glargine + 0 0 0 00.010 mg/ml of polysorbate 20 Insulin glargine + 0 0 0 0 0.015 mg/ml ofpolysorbate 20 Insulin glargine + 0 0 0 1 0.020 mg/ml of polysorbate 20Insulin glargine + 0 0 0 0 0.030 mg/ml of polysorbate 20

Storage for 6 Months at 5° C.

Number of vials with particle formation after Test sample 7 days 14 days21 days 28 days Insulin glargine 1 10 10 10 Insulin glargine + 0 0 0 10.010 mg/ml of polysorbate 20 Insulin glargine + 0 0 0 0 0.015 mg/ml ofpolysorbate 20 Insulin glargine + 0 0 0 1 0.020 mg/ml of polysorbate 20Insulin glargine + 0 0 1 0 0.030 mg/ml of polysorbate 20

Storage for 3 Months at 25° C.

Number of vials with particle formation after Test sample 7 days 14 days21 days 28 days Insulin glargine 9 10 10 10 Insulin glargine + 2 2 2 20.010 mg/ml of polysorbate 20 Insulin glargine + 0 0 0 1 0.015 mg/ml ofpolysorbate 20 Insulin glargine + 0 0 0 0 0.020 mg/ml of polysorbate 20Insulin glargine + 0 0 0 0 0.030 mg/ml of polysorbate 20

Storage for 6 Months at 25° C.

Number of vials with particle formation after Test sample 7 days 14 days21 days 28 days Insulin glargine 10 10 10 10 Insulin glargine + 0 0 0 10.010 mg/ml of polysorbate 20 Insulin glargine + 0 0 1 0 0.015 mg/ml ofpolysorbate 20 Insulin glargine + 0 0 0 0 0.020 mg/ml of polysorbate 20Insulin glargine + 0 0 0 0 0.030 mg/ml of polysorbate 20

Storage for 1 Month at 37° C.

Number of vials with particle formation after Test sample 7 days 14 days21 days 28 days Insulin glargine 0 10 10 10 Insulin glargine + 0 3 3 50.010 mg/ml of polysorbate 20 Insulin glargine + 0 0 0 0 0.015 mg/ml ofpolysorbate 20 Insulin glargine + 0 0 0 0 0.020 mg/ml of polysorbate 20Insulin glargine + 0 0 0 0 0.030 mg/ml of polysorbate 20

Storage for 3 Months at 37° C.

Number of vials with particle formation after Test sample 7 days 14 days21 days 28 days Insulin glargine 5 9 10 10 Insulin glargine + 1 1 1 10.010 mg/ml of polysorbate 20 Insulin glargine + 0 0 0 0 0.015 mg/ml ofpolysorbate 20 Insulin glargine + 0 0 0 0 0.020 mg/ml of polysorbate 20Insulin glargine + 0 0 0 0 0.030 mg/ml of polysorbate 20

Storage for 6 Months at 37° C.

Number of vials with particle formation after Test sample 7 days 14 days21 days 28 days Insulin glargine 10 10 10 10 Insulin glargine + 0 0 0 00.010 mg/ml of polysorbate 20 Insulin glargine + 0 0 1 0 0.015 mg/ml ofpolysorbate 20 Insulin glargine + 0 0 0 0 0.020 mg/ml of polysorbate 20Insulin glargine + 1 1 1 1 0.030 mg/ml of polysorbate 20

Without addition of polysorbate 20, particle formation can occur in thesolution even after 7 days in use. By addition of polysorbate 20, theparticle formation can be markedly suppressed during the in use period.

The stabilizing action of polysorbate 20 is retained even on storage atelevated temperatures for a period of 3 months.

A decline in the stabilizing action due to possible hydrolysis of thepolysorbate in the acidic medium of the solution cannot be determined incomparison with the data after storage for 1 month.

Example 2 Stabilization of Insulin Glargine Using Polysorbate 20 UnderPhysico-Mechanical Stress Loading

a) The solution is sterile-filtered through a combination of 0.2 μm und0.1 μm filters. It is then poured into 10 ml injection vials and sealedusing crimp caps having an inserted sealing disk.b) A comparison solution is prepared identically, but first a suitableamount of surfactant (0.010-0.030 mg/ml of polysorbate 20) is suspendedin water for injection.

The samples are stored at +5° C., 25° C. und 37° C. for a fixed periodof time. 5 samples in each case are then subjected to a shaking test.The results are shown in the table below, the limit 15 FNU correspondsto turbidities which are discernible in daylight.

Storage for 1 Month at 5° C.

Number of vials >15 FNU 0 0.5 1 2 3 4 6 Test sample days days day daysdays days days 8 days 10 days Insulin glargine 0 0 0 2 3 3 4 4 4 Insulinglargine + 0 0 0 0 0 1 3 4 5 0.010 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.015 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.020 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.030 mg/ml of polysorbate 20

Storage for 1 Month at 25° C.

Number of vials >15 FNU 0 0.5 1 2 3 4 6 Test sample days days day daysdays days days 8 days 10 days Insulin glargine 0 0 0 1 1 1 1 2 3 Insulinglargine + 0 0 0 0 0 0 1 2 3 0.010 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.015 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.020 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.030 mg/ml of polysorbate 20

Storage for 1 Month at 37° C.

Number of vials >15 FNU 0 0.5 1 2 3 4 6 Test sample days days day daysdays days days 8 days 10 days Insulin glargine 0 0 0 2 5 5 5 5 5 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.010 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.015 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.020 mg/ml of polysorbate 20 Insulinglargine + 0 0 0 0 0 0 0 0 0 0.030 mg/ml of polysorbate 20

Without addition of polysorbate 20, even after 2 days of severephysicomechanical stress, a visible turbidity can occur in the solution.By addition of polysorbate 20, the formation of turbidity duringphysicomechanical stressing can be markedly delayed. The stabilizingaction of polysorbate 20 is retained even on storage at elevatedtemperatures.

A decline in the stabilizing action due to possible hydrolysis of thepolysorbate in the acidic medium of the solution cannot be detected.

Example 3 Comparison of the Stabilization of the in Use Period ofInsulin Glargine Using Polysorbate 20 (Tween® 20) and Using Polysorbate80 (Tween® 20)

Open 10 vials in each case to give 5 ml of insulin glargine injectionsolution and

a) addition of 0.001 mg/ml of polysorbate 20b) addition of 0.01 mg/ml of polysorbate 20c) addition of 0.001 mg/ml of polysorbate 80d) addition of 0.01 mg/ml of polysorbate 80in the form of a concentrated stock solution.

The samples are then subjected to an in use test.

The results are shown in the table below.

Vials with particle formation after Test sample 7 days 14 days 21 days28 days Insulin glargine + no yes Yes, Yes, 0.001 mg/ml of particlesparticles polysorbate 20 increasingly increasingly occur occur Insulinglargine + no no no no 0.010 mg/ml of polysorbate 20 Insulin glargine +no yes Yes, Yes, 0.001 mg/ml of particles particles polysorbate 80increasingly increasingly occur occur Insulin glargine + no no no no0.010 mg/ml of polysorbate 80

An addition of polysorbate 20 or of polysorbate 80 in a concentration of0.001 mg/ml are equally able to stabilize the solution against particleformation during the in use period.

1. A pharmaceutical formulation comprising: (a) Gly(A21), Arg(B31),Arg(B32)-human insulin; (b) polysorbate 20; (c) sodium chloride; (d)glycerol; (e) m-cresol; and (f) water, wherein the pharmaceuticalformulation has a pH in the acidic range from 3.5 to 6.8.
 2. Thepharmaceutical formulation as claimed in claim 1, wherein the Gly(A21),Arg(B31), Arg(B32)-human insulin is present at a concentration of about1.4 to about 35 mg per milliliter.
 3. The pharmaceutical formulation asclaimed in claim 1, wherein the Gly(A21), Arg(B31), Arg(B32)-humaninsulin is present at a concentration of about 3.6 mg per milliliter. 4.The pharmaceutical formulation as claimed in claim 3, further includingzinc.
 5. The pharmaceutical formulation as claimed in claim 1, whereinpolysorbate 20 is present at a concentration of about 5 to 120 μg permilliliter.
 6. The pharmaceutical formulation as claimed in claim 1,wherein polysorbate 20 is present at a concentration of about 20 to 75μg per milliliter.
 7. The pharmaceutical formulation as claimed in claim1, wherein sodium chloride is present at a concentration of up to 150mM.
 8. The pharmaceutical formulation as claimed in claim 1, whereinglycerol is present at a concentration of about 100 to 250 mM.
 9. Thepharmaceutical formulation as claimed in claim 1, further comprising abuffer.
 10. The pharmaceutical formulation as claimed in claim 9,wherein the buffer is chosen from TRIS, phosphate, citrate, acetate, andglycylglycine.
 11. The pharmaceutical formulation as claimed in claim10, wherein said buffer is present in a concentration of 5-250 mM. 12.The pharmaceutical formulation as claimed in claimed 1, wherein thepharmaceutical formulation has a pH in the acidic range from 3.5 to 4.5.13. The pharmaceutical formulation as claimed in claimed 1, wherein thepharmaceutical formulation has a pH of 4 (+/−0.2).